Chromosomes are structures, which contain genes and, thereby, hereditary information. They contain DNA which is packed with proteins and are to be found in the nuclei of eukaryotic cells.
In the middle of the 20th century techniques were developed in order to analyze the number and structure of cells which were metaphasing. In a metaphase preparation the chromosomes of a cell lie spread out beside one another, i.e. spread out on a microscope slide so that they can be counted under the microscope and compared with one another. In good preparations the individual chromosomes have the frequently displayed X-like shape.
A classic staining technique to display metaphase chromosomes is the Giesma staining. After the chromosomes have been treated with the enzyme trypsin the chromatin complex of DNA and bound histon proteins is stained with Methylene Blue. Depending upon the base composition, chromatin condensation and the time-point of the replication alternate dark-colored sections (G-bands) and light-colored sections (R-bands) are formed. The banded profile makes it possible to obtain an unambiguous identification of all the chromosomes in humans and some animals (Zhao et al., 1998).
Other analytical methods for chromosomes which are based upon staining techniques are fluorescence in situ hybridisation (FISH) and spectral karyotyping (SKY).
In the FISH technique artificially-produced probes of nucleic acids are employed which hybridize by base pairing to the nucleic acid to be detected. After hybridization, which takes from 1 hour to several days, hybrid molecules made from the nucleic acids of the preparation and the probe are present. The bound probe molecules can be detected. With an indirect marking, the detection is done using an antibody stain or Avidin which in turn are bound to fluorochromes. For each fluorochrome, individual pictures are taken using filter systems which are then superimposed (Bayani und Squire, 2002; Zhao et al., 1998).
The SKY technique is based upon Fourier Spectroscopy in which an interferometer allows the measurement of the entire emitted light spectrum for each pixel. To facilitate this, chromosome specific color probes are employed so that each chromosome receives a characteristic emission spectrum. By converting these specific spectra into pseudo colors it is possible to identify each chromosome and, if appropriate, chromosomal aberrations (Bayani und Squire, 2002).
In the analysis of stained chromosomes, the color quality, the degree of condensation and the spread determine the banded profile or the spectral properties of the chromosomes. In this way, the significance of these techniques for possible structural anomalies of the chromosomes varies depending upon the staining and preparation methods. A further disadvantage of the staining techniques is the time intensive nature of their execution and analysis which require special methodical skill and a precise knowledge of the banding of chromosomes. Furthermore repeated staining or double staining, such as, for example, FISH on Giemsa is often necessary to allow analysis to be carried out and verified.
In addition to the techniques based on staining described above, atomic force microscopy uses unmarked chromosomes. In this case karyotypization is carried out based on the length, width, height or volume or the specific height profile of each chromosome as a microscopically small needle scans over the surface of the sample. Technically this method is extremely demanding and highly sensitive to disturbance by vibration, heat and static loading. Furthermore it exhibits system generated faults so that rather than producing an image of the actual surface of the sample an image of the folding of the geometry of the needle point with the structure of the surface is produced.
With the help of chromosome analysis, variations in the karyotype, i.e. changes of the chromosome number, such as, for example trisomy 21, monosomy X, or structural aberrations such as, for example, translocation, deletion, inversion can be detected. In diagnostics a distinction is made between prenatal and postnatal chromosome analysis as well as pre-implantation analysis. By means of prenatal karyotyping it is possible to examine fetuses during pregnancy following an amniocentesis or a chorionic villus biopsy for possible diseases and damages. Particularly in the field of prenatal diagnostics there is a great need for a quick and reliable test for karyotyping which can also be carried out by non-specialist personnel working in a physician's practice. Because of the increasing age of expectant parents prenatal diagnostics have earned an increasing level of importance.
Until now, in the prior art a chromosome analysis is carried out solely by disturbance-prone, elaborate atomic force techniques or by staining techniques which are time consuming and depend upon staining quality, degree of condensation and spreading of the chromosomes. Furthermore, an analysis based upon staining techniques requires a precise knowledge of chromosome banding.
The present invention, therefore, faces the problem of providing a quick and simple method for improved chromosome analysis which is independent of marking or staining techniques.
According to the invention this problem is solved by a method for analyzing chromosomes. In the method according to the invention a chromosome preparation is made and its interference properties are measured, thereby characterizing chromosome structures.